JP7179041B2 - Trifunctional T cell-antigen couplers and methods and uses thereof - Google Patents

Description

RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61/936,906, filed February 7, 2014, the contents of which are hereby incorporated by reference in their entirety. .

The present invention relates to methods of treating cancer by engineering T cells that have increased cytotoxicity towards specific target cells and reduced off-target toxicity. In particular, the present disclosure relates to engineering T cells to express novel biological factors that mimic naturally occurring T cell activation processes.

Cancer is a serious health challenge, with more than 150,000 cases of cancer expected to be diagnosed in Canada in 2013 alone. While patients with early-stage disease can be effectively treated with conventional therapies (surgery, radiotherapy, chemotherapy), patients with advanced disease have fewer options available, and these options are usually substantive. It is palliative. Active immunotherapy seeks to harness the patient's immune system to clear tumor deposits and offers an exciting option for patients who have failed conventional therapies (Non-Patent Document 1). In fact, several clinical studies have demonstrated that immunotherapy with T cells can be curative for patients with advanced melanoma, supporting the usefulness of this approach (Non-Patent Literature). 1). In addition, patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) have also been effectively treated and cured with T-cell immunotherapy. (Non-Patent Document 2) (Non-Patent Document 3). Although there are several immunotherapeutic approaches, engineering T cells with chimeric receptors allows any patient's immune cells to become major histocompatibility complex (MHC)-independent to any desired immunotherapeutic approach. It is possible to target the target of So far, the chimeric receptors used to engineer T cells consist of a targeting domain, usually a single-chain fragment variable (scFv); a transmembrane domain; is composed of a cytoplasmic domain containing the signaling elements of (Non-Patent Document 4). Such chimeric receptors have been called "T-bodies", "Chimeric Antigen Receptors (CAR)" or "Chimeric Immune Receptors (CIR)". - Currently, many researchers use the term "CAR" (Non-Patent Document 4). Such CARs have been considered in terms of elements, and scientists have spent considerable time investigating the effects of various cytoplasmic signaling domains on CAR function. First generation CARs used a single signaling domain from CD3ζ or FcεRIγ. Second-generation CARs combined the signaling domain of CD3zeta with the cytoplasmic domains of co-stimulatory receptors from the CD28 or TNFR families of receptors (Non-Patent Document 4). Third-generation CARs have multiple co-stimulatory domains, but there is concern that third-generation CARs may lose antigen specificity (Non-Patent Document 5). Many CAR-engineered T cells being clinically tested use second-generation CARs in which CD3ζ is coupled to the cytoplasmic domain of CD28 or CD137 (Non-Patent Document 5) (Non-Patent Document 6) (Non-Patent Document 6). Patent document 7).

CAR-engineered T cells have shown considerable promise in clinical applications, but rely on synthetic methods to replace activation signals provided by the T cell receptor (TCR). Since this synthetic receptor does not deliver all of the signaling components associated with the TCR (e.g., CD3 epsilon, Lck), whether T cells are optimally activated by CAR, or whether CAR activation is associated with TCR It is still unclear how it affects cell differentiation (eg development or memory). Furthermore, since the CAR signaling domain is separated from its natural regulatory partner by the very nature of the CAR structure, there is also an inherent risk that CAR could lead to low levels of constitutive activation that could lead to off-target toxicity. be.

Given these limitations, it is preferable to redirect T cells to attack tumors via their native TCR. To that end, a class of recombinant proteins called "Bispecific T-cell Engagers" (BiTEs) has been generated (8) (9). These proteins use bispecific antibody fragments to cross-link T cell TCR receptors with target antigens. This results in efficient T cell activation and causes cytotoxicity. Similarly, bispecific antibodies have been generated that achieve this goal, and some scientists simply use chemical conjugation to link an anti-CD3 antibody to a tumor-specific antibody. 8). Although such bispecific proteins have shown some activity in vitro, GMP manufacturing, short biological half-life and bioavailability are major challenges for the successful use of these molecules in cancer treatment. is throwing Furthermore, these molecules are also unable to properly recapitulate naturally occurring TCR signaling as they do not engage the TCR co-receptors (CD8 and CD4).

Therefore, there remains a need for T cell-antigen couplers with enhanced activity and safety compared to conventional CARs.

Humphries, C.I. (2013), Adoptive cell therapy: Honing that killer instinct., Nature 504:S13-5. Fry, T.; J. , Mackall, C.I. L. (2013), "T-cell adoptive immunotherapy for acute lymphoblastic leukemia," Hematology American Society Hematology Education Program (Hematology Am. Soc. Hematol. Ed.). .Program) 2013, 348-353. Kochenderfer, J.; N. , Rosenberg, S.; A. (2013), "Treatment B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors," Nature Reviews Clinical Oncology. (Nat. Rev. Clin. Oncol.), 10:267-276. Dotti, G.; , Savoldo, B. , Brenner, M.; (2009), "15 Years of Chimeric Antigen Receptor-Based Gene Therapy: 'Still Close to the Goal? "(Fifteen years of gene therapy based on chimeric antibody receptors: "are we almost there yet?")", Hum. Gene Ther. 20:1229-1239. Han, E. Q. , Li, X. and Han, S.; (2013), "Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges," Journal of Hematology and Oncology. (J. Hematol. Oncol.), 6:47. Finney, H. M. , Akbar, A.; N. , Lawson, A.; D. G. (2004), Activation of resting human primary T cells with chimeric receptors: co-stimulation from CD28, inducible co-stimulators, CD134 and CD137 contiguous with signals from the TCR zeta chain. T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain)", Journal of Immunology (J. Milone, M.; C. , Fish, J.; D. , Carpenito, C.I. , Carroll, R. G. , Binder, G.; K. , Teachey, D. , Samanta, M.; , Lakhal, M.; , Gross, B. Danet-Desnoyers, G.I. (2009), "Chimeric receptors containing CD137 signal transduction domains mediate enhanced in vivo survival of T cells and enhanced anti-leukemia effects." survival of T cells and increased antileukemia efficacy in vivo).", Molecular Therapy (Mol. Ther.) 17:1453-1464. Chames P.S. and Baty, D.M. (2009), "Bispecific antibodies for cancer therapy: the light at the end of the tunnel?", Monoclonal Antibodies ( MAbs) 1:539-547. Portell, C.; a. , Wenzell, C.I. M. , Advani, A.; S. (2013), "Clinical and pharmacologic aspects of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia", Clinical Pharmacology. Pharmacol.) 5:5-11.

We have found that trifunctional T-cell-antigen couplers that better mimic naturally occurring signaling through the T-cell receptor (TCR) while maintaining non-main histocompatibility complex targeting. It has been shown to exhibit higher activity and safety than conventional chimeric antigen receptors.

Therefore, one aspect of the present disclosure is
a. a first polynucleotide encoding a target-specific ligand;
b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex and c. A nucleic acid is provided that includes a third polynucleotide that encodes a T-cell receptor signaling domain polypeptide.

Another aspect of the disclosure provides polynucleotides encoded by the above nucleic acids.

Another aspect of the disclosure provides an expression vector comprising the nucleic acid described above.

Yet another aspect of the disclosure provides T cells expressing the above nucleic acids. Another aspect of the disclosure provides a pharmaceutical composition comprising the T cells and a carrier.

Also disclosed is the use of T cells to treat cancer in a subject in need thereof, said T cells comprising: a. a first polynucleotide encoding a target-specific ligand;
b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex and c. Use is provided to express a nucleic acid comprising a third polynucleotide encoding a T-cell receptor signaling domain polypeptide.

In one embodiment, said target-specific ligand binds to an antigen on cancerous cells.

In another embodiment, said target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide or scFv.

In another embodiment, the protein associated with the TCR complex is CD3.

In another embodiment, the ligand that binds the protein associated with said TCR complex is a single chain antibody.

In another embodiment, the ligand that binds to a protein associated with said TCR complex is UCHT1 or a variant thereof.

In another embodiment, said T-cell receptor signaling domain polypeptide comprises a cytoplasmic domain and a transmembrane domain.

In another embodiment, said cytoplasmic domain is the CD4 cytoplasmic domain and the pre-transmembrane domain is the CD4 transmembrane domain.

In another embodiment, said first polynucleotide and third polynucleotide are fused to said second polynucleotide.

In another embodiment, said second polynucleotide and third polynucleotide are fused to said first polynucleotide.

This disclosure also provides
a. a first polynucleotide encoding a target-specific ligand;
b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex;
c. a third polynucleotide encoding a T-cell receptor signaling domain polypeptide and d. Vector constructs containing promoters capable of functioning in mammalian cells are provided.

In one embodiment, said first polynucleotide and said third polynucleotide are fused to said second polynucleotide to form a T cell antigen coupler fusion, wherein the coding sequence of said T cell antigen coupler fusion is operably linked to said promoter.

In another embodiment, said second polynucleotide and said third polynucleotide are fused to said first polynucleotide to form a T cell antigen coupler fusion, wherein said T cell antigen coupler fusion coding sequence comprises: is operably linked to said promoter.

The disclosure also provides isolated T cells transfected with the vector constructs.

Other features and advantages of the present disclosure will become apparent from the detailed description below. However, it will be apparent to those skilled in the art from this detailed description that various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art. It should be understood that, while indicating preferred embodiments, they are given by way of illustration only.

Schematic overview of a trifunctional T cell-antigen coupler (Tri-TAC) compared to a conventional second generation CAR. FIG. 4 shows the results of surface expression analysis of Tri-TAC mutants and classical CAR. FIG. 2 shows results of cell activation analysis focusing on various markers IFN-γ, TNF-α and CD107a. FIG. 2 shows the results of a killing analysis of two different cell lines expressing (D2F2E2) or not (D2F2), the molecular targets of classical CAR and Tri-TAC. FIG. 1 shows spontaneous initiation of T cell activation (A), currently used artificial methods of T cell activation (B and C) and TAC activation techniques (D). (A) Form 1 of TAC molecule and (B) Form 2 of TAC molecule. FIG. 3 shows the functionality of scFv CD4 TAC. (A) Histograms showing surface expression of scFv CD4 TAC receptor compared to empty vector and (B) antigen-specific activity of T cells expressing scFv CD4 TAC (top) or scFv CAR (bottom). (C) shows comparable killing of MCF-7 human tumor cell line (Her2 positive) by scFv CD4 TAC and scFv CAR. FIG. 2 shows the properties of CD4-TAC Form 2. FIG. (A) is a histogram showing surface expression of the darpin CD4 TAC receptor compared to empty vector and (B) shows cytokine production and degranulation of darpin TAC form 2 engineered T cells exposed to Her2 antigen. , (C) shows proliferation of CD4 TAC form 2 compared to empty vector controls. FIG. 2 shows the functionality of Darpin CD4 TAC Form 1. FIG. (A) shows surface expression of darpin CD4 YAC compared to darpin CAR and control NGFR alone, (B) shows proliferation of CD4 TAC structure 1, (C) and (D) show different activation and deactivation. Percentage of cells positive for granule markers is shown. FIG. 1 shows cytotoxicity and general activity of TAC and CAR. TAC, CAR or empty vector control engineered cells were incubated in various human tumor cell lines. FIG. 2 shows receptor surface expression and activation of various TAC controls. (A) shows cell surface expression (left), degranulation (middle) and cytokine production (right) and (B) shows that only full-length CD4-TAC can elicit a cytotoxic response. FIG. 1 shows properties of various transmembrane TAC mutants. (A) is an overview of the various transmembrane domain constructs, (B) shows the expression surface of the various constructs engineered in CD8-purified T cells, and (C) shows the various transmembrane domain constructs for degranulation and cytokine production. shows the test results for the mutants of FIG. 3 shows the interaction of Lck with TAC mutants. (A) shows the ability of full-length TAC and cytoplasmic defects to disrupt Lck, and (B) is a densitometric analysis of Lck detected in the pellet of (A). CD4 TAC surface expression and activity compared to BiTE-like mutants. (A) shows the surface expression of control NGFR alone, CD4 TAC and BiTE-like mutants, and (B) a comparison of cytotoxicity in various cell lines. FIG. 3 shows wild-type CD4 TAC compared to a random mutagen library of UCHT1. (A) shows a schematic representation of the mutants, (B) is a histogram showing the surface expression of the library, and (C) shows the ability of the library to activate T cells and produce cytokines. FIG. 4 shows enhanced surface expression of A85V, T161P mutants. (A) Final CD/CD8 population comparison between CD4 TAC and A85V, T161P mutants, (B) showing enhanced surface expression of A85V, T161P mutants, (C) A85V, Figure 2 shows decreased cytokine production in the T161P mutant. Cytotoxicity and proliferation of A85V, T161P mutants. (A) shows the cytotoxicity of A85V, T161P mutants in various cell lines and (B) shows cell growth in culture over 2 weeks.

(i) Definitions As used herein, the term "cell" includes single cells and multiple cells.

As used herein, the term "T cells" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells, also called T lymphocytes, can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of a T cell receptor (TCR) on their cell surface. There are several subsets of T cells with distinct functions, including T helper cells, cytotoxic T cells, memory T cells, regulatory T cells and natural killer T cells. It is not limited to these.

As used herein, the term "T cell antigen coupler" refers to an engineered nucleic acid construct or polypeptide that, when expressed on a T cell, targets a specific antigen for delivery to that T cell. pointing.

The terms "polynucleotide" and/or "nucleic acid sequence" and/or "nucleic acid" as used herein refer to a series of nucleoside or nucleotide monomers composed of bases, sugars and intersugar (backbone) linkages. pointing. The term also includes modified or substituted sequences or portions thereof, including non-naturally occurring monomers. The nucleic acid sequences of the present application can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. Such sequences may also contain modified bases. Such modified bases include, for example, aza and deazaadenine, guanine, cytosine, thymidine and uracil and xanthine and hypoxanthine. Nucleic acids of the present disclosure can be isolated from biological organisms formed by genetic recombination laboratory methods or obtained by chemical synthesis or other known protocols for making nucleic acids.

The terms "isolated polynucleotide" or "isolated nucleic acid sequence", as used herein, refer to cellular material or culture medium when produced by recombinant DNA techniques, or chemical sequence when chemically synthesized. It refers to a nucleic acid that is substantially free of chemical precursors or other chemicals. Also, an isolated nucleic acid is substantially free of the sequences that originally flank the nucleic acid (ie, sequences at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA, and can be double-stranded or single-stranded, and represents the sense or antisense strand. Furthermore, the term "nucleic acid" includes its complementary nucleic acid sequence.

The terms "recombinant nucleic acid" or "engineered nucleic acid," as used herein, refer to nucleic acids or polynucleotides that do not exist in a biological organism. For example, a recombinant nucleic acid can be formed by genetic recombination laboratory methods (such as molecular cloning), resulting in a sequence that would otherwise not exist in nature. Recombinant nucleic acids can also be made by chemical synthesis or other known protocols for making nucleic acids.

The terms "polypeptide" or "protein" as used herein refer to a chain of amino acids corresponding to those encoded by a nucleic acid. Polypeptides or proteins of the present disclosure can be peptides, typically describing chains of amino acids from 2 to about 30 amino acids. The term protein, as used herein, also describes amino acid chains having more than 30 amino acids and can be fragments or domains of proteins or full-length proteins. Additionally, the term protein as used herein can refer to a linear chain of amino acids or it can refer to a chain of amino acids that has been processed or folded into a functional protein. However, 30 is an arbitrary number for distinguishing between peptides and proteins, and these terms can be used interchangeably with respect to amino acid chains. A protein of the disclosure may be obtained by isolation and purification of the protein from cells in which it is produced naturally by enzymatic (proteolytic) cleavage and/or recombinantly by expression of a nucleic acid encoding the protein or fragment of the disclosure. Obtainable. Proteins and/or fragments of the present disclosure may also be obtained by chemical synthesis or other known protocols for producing proteins and fragments.

The term "isolated polypeptide" refers to a polypeptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques or chemical precursors or other chemicals when chemically synthesized. It means

The term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, single chain antibodies, chimeric antibodies and antibody fusions. The antibody may be from a recombinant source and/or produced in a transgenic animal. The term "antibody fragment" as used herein includes Fab, Fab', F(ab') ₂ , scFv, dsFv, ds-scFv, dimers, minibodies, diabodies and multimers thereof, multispecific Including, but not limited to, antibody fragments as well as domain antibodies.

As used herein, the term "vector" refers to a polynucleotide that can be used to deliver a nucleic acid inside a cell. In one embodiment, the vector is an expression vector that includes an expression control sequence (eg, promoter) operably linked to the nucleic acid to be expressed intracellularly. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids and viruses.

(ii) Compositions We have developed trifunctional T cell-antigen couplers that better mimic naturally occurring signaling through the T cell receptor (TCR) while maintaining MHC-unrestricted targeting. (Tri-TAC) was developed. Specifically, we generated molecules in which the transmembrane and intracellular regions of the CD4 co-receptor were fused to a single-chain antibody that binds CD3, which localize to lipid rafts and bind Lck, respectively. did. This construct is designed to bring CD3 molecules and the TCR into the region of lipid rafts, bringing Lck into the vicinity of the TCR, similar to naturally occurring MHC binding. To target this chimeric receptor, designed ankyrin repeats (darpin) were linked to the CD4-UCHT1 chimera to create a trifunctional T cell-antigen coupler (Tri-TAC).

Experimentally, human T cells were engineered to express prototype Tri-TAC or conventional CAR with the same darpin. As a result, it was revealed that Tri-TAC-manipulated T cells exhibited functionality equivalent to that of conventional CAR in all respects. For two parameters (TNF-α production and CD107a mobilization) Tri-TAC was found to be more active than conventional CAR. Furthermore, the data show that Tri-TAC per molecule shows a significant enhancement of activity. Furthermore, Tri-TAC exhibits improved safety compared to conventional CARs, as the activation domain is not part of this protein.

Therefore, the present disclosure provides
a first polynucleotide encoding a target-specific ligand;
A nucleic acid comprising a second polynucleotide encoding a ligand that binds to the TCR complex and a third polynucleotide encoding a T-cell receptor signaling domain polypeptide.

In one embodiment, the nucleic acid is a recombinant, ie engineered nucleic acid. In another embodiment, the first, second and/or third polynucleotides are recombinant, ie engineered polynucleotides.

The disclosure also relates to polypeptides encoded by the nucleic acids and compositions comprising the nucleic acids.

A nucleic acid comprising each of the first, second and third polynucleotides and the polypeptide encoded by this nucleic acid is also referred to herein as a trifunctional T cell-antigen coupler or Tri-TAC.

Target-Specific Ligands Target-specific ligands direct T cell-antigen couplers to target cells. A target-specific ligand therefore refers to any substance that binds directly or indirectly to a target cell. A target cell can be any cell associated with a disease state, including but not limited to cancer. In one embodiment, a target-specific ligand binds to an antigen (a protein produced by a cell capable of eliciting an immune response) on a target cell. This target-specific ligand can also be called an antigen-binding domain.

In one embodiment, the target cells are tumor cells. In this case, the target-specific ligand can bind to tumor antigens or tumor-associated antigens on tumor cells. Tumor antigens are well known in the art. As used herein, the term "tumor antigen" or "tumor-associated antigen" refers to any antigenic antigen produced in tumor cells that elicits an immune response in the host (which can be represented, for example, by MHC complexes). means substance. Tumor antigens, when proteinaceous, include, for example, eight or more amino acids up to the full protein, and at least one antigenic fragment of the full-length protein that can be represented as an MHC complex. It can be a sequence of any number of amino acids between proteins. Examples of tumor antigens include HER2 (erbB-2), B-cell maturation antigen (BCMA), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125. , MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), prostate-specific antigen (PSA), glioma-associated antigen, β- human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO- 1, LAGE-1a, p53, protein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growth factor ( IGF (insulin growth factor)-I, IGF-II, IGF-I receptor and mesothelin, but not limited thereto.

Target-specific ligands include, for example, antibodies and fragments thereof, such as single-chain antibodies such as scFv, or small proteins that bind to target cells and/or antigens.

One example of a target-specific ligand is an engineered ankyrin repeat (darpin) that targets specific cells and/or antigens. In one embodiment, the target-specific ligand is darpin, which targets HER2 (erbB-2). One example of a darpin that targets HER2 (erB-2) is shown as SEQ ID NOS: 7 and 8 herein.

Another example of target-specific ligands are scFvs targeted to specific cells and/or antigens. In one embodiment, the target-specific ligand is an scFv that binds HER2 (erB-2). One example of a scFv that binds HER2 (erB-2) is shown as SEQ ID NOS: 22 and 23 herein.

Ligands that bind to the TCR complex T cell-antigen couplers have been designed to recruit the T cell receptor (TCR) in combination with co-receptor stimulation. Thus, this T cell-antigen coupler contains a ligand that binds to a protein associated with the T cell receptor complex.

The TCR (T cell receptor) is a complex of integral membrane proteins involved in the activation of T cells in response to antigen binding. The TCR consists of highly variable alpha (α) and beta (β) chains that are normally expressed as part of a complex with an invariant CD3 (cluster of differentiation 3) chain molecule. It is a disulfide-bonded membrane-anchored heterodimer. T cells that express this receptor are called α:β (or αβ) T cells, but a minority of T cells are alternatives formed by variable gamma (γ) and delta (δ) chains called γδ T cells. express the receptor. CD3 is a protein complex composed of four distinct chains. In mammals, this complex includes a CD3 gamma chain, a CD3 delta chain and two CD3 epsilon chains.

As used herein, the term "ligand that binds to a protein associated with the T-cell receptor complex" includes any substance that directly or indirectly binds to a protein of the TCR. Proteins associated with the TCR include, but are not limited to, the TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3 γ chain, CD3 δ chain and CD3 ε chain. is not limited to In one embodiment, the ligand that binds to a protein associated with the T cell receptor complex is TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, the CD3 delta chain and/or the CD3 epsilon chain.

In one embodiment, the ligand is an antibody or fragment thereof that binds CD3. Examples of CD3 antibodies are well known in the art (eg, muromonab, otelixizumab, teplizumab and visilizumab). In one embodiment, the antibody that binds CD3 is a single chain antibody, eg, a single chain variable fragment (scFv).

Another example of a CD3 antibody is UCHT1, which targets CD3ε. The sequences of UCHT1 are presented as SEQ ID NOS: 13 and 14 herein.

T-Cell Receptor Signaling Domain Polypeptides T-cell-antigen couplers comprise T-cell receptor signaling domain polypeptides. As used herein, the term "T cell receptor signaling domain" refers to a polypeptide that (a) localizes to lipid rafts and/or (b) binds Lck. A T-cell receptor signaling domain polypeptide can comprise one or more protein domains including, but not limited to, a cytoplasmic domain and/or a transmembrane domain. As used herein, "protein domain" refers to a conserved portion of a given protein sequence structure that can function and exist independently of the rest of the protein chain. In one embodiment, said T cell receptor signaling domain polypeptide comprises a cytoplasmic domain. In another embodiment, the T cell receptor signaling domain polypeptide comprises a transmembrane domain. In another embodiment, the T-cell receptor signaling domain polypeptide includes both a cytoplasmic domain and a transmembrane domain.

T-cell receptor signaling domain polypeptides include TCR co-receptors and costimulators and TCR co-receptors and costimulator protein domains.

"TCR co-receptor" refers to a molecule that assists the T cell receptor (TCR) in interacting with antigen presenting cells. TCR co-receptors include, for example, CD4, CD8, CD28, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CDt37 and CD154, but these is not limited to

"TCR co-stimulatory factor" refers to a molecule that is involved in a T-cell response to an antigen. Examples of TCR co-stimulatory factors include PD-1, ICOS, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, lymphocyte fiction associated antigen 1 (LFA-1), CD2, CD7, LIGHT, Ligands that specifically bind to NKG2C, B7-H3, and CD83 include, but are not limited to.

In one embodiment, a T-cell receptor signaling domain polypeptide comprises both the cytoplasmic and transmembrane domains of a TCR co-receptor or costimulator protein. These cytoplasmic and transmembrane domains can be from the same co-receptor or co-stimulatory factor or from different co-receptors or co-stimulatory factors. These cytoplasmic and transmembrane domains are optionally connected by a linker.

In one embodiment, the T cell receptor signaling domain polypeptide comprises the transmembrane and cytoplasmic domains of the CD4 co-receptor (see, eg, SEQ ID NOS: 17 and 18).

In another embodiment, the T-cell receptor signaling domain polypeptide comprises transmembrane and cytoplasmic domains of the CD8α co-receptor.

In other embodiments, the cytoplasmic and/or transmembrane domains of the T-cell receptor signaling domain polypeptide are synthetic. For example, the transmembrane domain is optionally a synthetic highly hydrophobic membrane domain.

In another example, the transmembrane domain is a glycophorin transmembrane domain. In yet another example, the T cell receptor signaling domain polypeptide includes a CD48 GPI signaling sequence for attaching the T cell antigen coupler to the membrane using a GPI anchor.

In addition to the three components of the T-cell antigen coupler described herein (target-specific ligand, ligand that binds the TCR complex, and T-cell receptor signaling domain polypeptide), other polypeptides can also be included. It is thought that it may be. For example, T cell antigen couplers optionally include additional polypeptides that serve to directly or indirectly target or activate T cells.

Linkers The various components of the T cell antigen coupler can be directly fused together or they can be joined by at least one linker, optionally a peptide linker. This peptide linker can be of any size as long as it does not interfere with the function of the individual binding moieties. In one embodiment, the peptide linker is about 1 to 15 amino acids in length, more specifically about 1 to about 10 amino acids, most specifically about 1 to about 6 amino acids in length.

Linkers useful in T cell antigen couplers include, for example, _{the G4S3} linker. Other examples of linkers are peptides corresponding to SEQ ID NOs: 11, 12, 15, 16, 19, 20 and 21 and variants and fragments thereof.

Forms T cell-antigen couplers can exist in a variety of forms, as will be readily appreciated by those skilled in the art.

In one embodiment, a target-specific ligand and a T-cell receptor signaling domain polypeptide are both fused to a ligand that binds to the TCR complex. For example, the N-darpin TACs described herein (of SEQ ID NOs: 1 and 2, also referred to as Form 1) are in sequence:
i) N-darpin Tri TAC leader sequence (secretion signal) (SEQ ID NOS: 5 and 6)
ii) Darpin specific for Her2 antigen (SEQ ID NOs: 7 and 8)
iii) Myc tag (SEQ ID NOS: 9 and 10)
iv) Linker 1 (SEQ ID NO: 11 and 12)
v) UCHT1 (SEQ ID NOS: 13 and 14)
vi) Linker 2 (SEQ ID NO: 15 and 16)
vii) CD4 (SEQ ID NO: 17 and 18)
including.

In another embodiment, darpin is replaced with scFv ScFv (SEQ ID NOS:22 and 23) specific for the Her2 antigen.

In another embodiment, the ligand that binds the TCR complex and the T cell receptor signaling domain polypeptide are both fused to a target-specific ligand (of SEQ ID NOS:3 and 4, also referred to as Form 1). Alternative forms should be readily apparent to those skilled in the art.

Vector Constructs A variety of delivery and expression vectors can be used to introduce the nucleic acids described herein into cells. Thus, optionally including a polynucleotide as described above in a vector results in a vector construct, sometimes referred to herein as a vector.

Accordingly, the present disclosure
a. a first polynucleotide encoding a target-specific ligand;
b. a second polynucleotide encoding an antibody that binds to CD3 and c. It also relates to a vector comprising a third polynucleotide encoding a T-cell receptor signaling domain polypeptide and, optionally, a promoter capable of functioning in mammalian cells.

Promoters, which are regions of DNA that initiate transcription of a particular nucleic acid sequence, are well known in the art. A "promoter capable of functioning in mammalian cells" refers to a promoter that drives expression of an associated nucleic acid sequence in mammalian cells. A promoter that drives expression of a nucleic acid sequence can be said to be "operably linked" to that nucleic acid sequence.

In one embodiment, a first polynucleotide and a third polynucleotide are fused to said second polynucleotide to form a T cell antigen coupler fusion, the T cell antigen coupler fusion coding sequence operating with a promoter. connected as possible.

In another embodiment, the second polynucleotide and the third polynucleotide are fused to the first polynucleotide to form a T cell antigen coupler fusion, the T cell antigen coupler fusion coding sequence operating with the promoter. connected as possible.

Optionally, the vector is designed to be expressed in mammalian cells such as T cells. In one embodiment, the vector is a viral vector, optionally a retroviral vector.

Vectors that are useful include those derived from lentiviruses, Murine Stem Cell Viruses (MSCV), poxviruses, oncoretroviruses, adenoviruses and adeno-associated viruses. Other delivery viruses that are useful include vectors derived from herpes simplex virus, transposons, vaccinia virus, human papilloma virus, simian immunodeficiency virus, HTLV, human foamy virus and variants thereof. Additional vectors that are useful include those derived from spumaviruses, mammalian B-type retroviruses, mammalian C-type retroviruses, avian C-type retroviruses, mammalian D-type retroviruses, and HTLV/BLV-type retroviruses. One example of a lentiviral vector useful in the compositions and methods of this disclosure is the pCCL vector.

Polynucleotide and Polypeptide Modifications Many modifications can be made to the polynucleotide sequences, including vector sequences, and to the polypeptide sequences disclosed herein, which will be apparent to those skilled in the art. Modifications include nucleotide or amino acid substitutions, insertions or deletions, or changes in the relative position or order of nucleotides or amino acids.

In one embodiment, the polynucleotides described herein are modified or mutated to optimize the function of its encoded polypeptide and/or its T cell antigen coupler function, activity and/or expression. can be done.

It is shown herein that UCHT1 mutants can be generated that result in enhanced surface expression of TAC (Figures 15-17). Thus, in one embodiment, the TAC is a modified or mutated ligand that binds to the TCR complex, and the TAC comprising the modified or mutated antibody is a wild-type, i.e. unmodified or mutated ligand that binds to the TCR complex containing ligands that have increased surface expression and/or activity compared to TAC containing type ligands. One example of a mutated or modified antibody that binds to CD3 is the UCHT1 A85V, T161P mutant described herein (SEQ ID NOs:24 and 25).

Sequence Identity Polynucleotides of the present application are at least about 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical to the nucleic acid molecules of the present application at least 97% identity, at least 98% identity, or at least 99% or 99.5% identity. Also, "Polypeptides of the present application are at least about 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, to a polypeptide of the present application. % identity, at least 97% identity, at least 98% identity, or at least 99% or 99.5% identity (or fragments thereof). , refers to the similarity of two nucleotide or polypeptide sequences that are aligned for best order matching. Identity is calculated according to methods known in the art. For example, if a nucleotide sequence (referred to as "sequence A") has 90% identity to a portion of SEQ ID NO: 1, then sequence A represents 100 nucleotide sequences of this reference portion of SEQ ID NO: 1. SEQ ID NO: 1, except that it can contain up to 10 points of mutation (such as substitutions with other nucleotides) per nucleotide of SEQ ID NO:1.

Preferably, the sequence identity is at least about 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, to the nucleotide sequences presented herein and/or their complementary sequences. % identity, at least 96% identity, at least 97% identity, at least 98% identity, or most preferably at least 99% or 99.5% identity. Sequence identity is also preferably at least about 70% identical, at least 80% identical, at least 90% identical, at least 95% identical to the polypeptide sequences set forth herein , at least 96% identity, at least 97% identity, at least 98% identity, or most preferably at least 99% or 99.5% identity. Sequence identity is preferably calculated using the GCG program from Bioinformatics (University of Wisconsin). Also, other programs for calculating sequence identity such as the Clustal W program (preferably using default parameters; Thompson, JD et al., Nucleic Acid Res. 22:4673-4680). Programs are also available.

Hybridization This application provides sequences having sufficient identity (hybridization techniques are well known in the art) to hybridize under stringent hybridization conditions to the nucleic acid molecules described herein. contains DNA with The present application also includes nucleic acid molecules that hybridize to one or more of the sequences described herein and/or their complementary sequences. Such nucleic acid molecules are preferably prepared under high stringency conditions (Sambrook et al., Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory). • Hybridize under a press (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). A high stringency wash is preferably low salt (preferably about 0.2% SSC) and a temperature of about 50-65° C., optionally performed for about 15 minutes.

Expression in T Cells T cell antigen couplers are designed to be expressed in T cells. Accordingly, one aspect of the present disclosure provides T cells expressing T cell antigen couplers. Another aspect of the present disclosure relates to T cells transduced or transfected with a T cell antigen coupler or a vector containing a T cell antigen coupler. Optionally, the T cells are isolated T cells.

T cells can be obtained from many sources, including blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, umbilical cord blood, thymus tissue, from sites of infection. Tissues include, but are not limited to, spleen tissue and tumors. In one embodiment, the T cells are autologous T cells. In another embodiment, the T cells are obtained from a T cell line. Methods for culturing and maintaining T cells in vitro are well known in the art.

The T cells obtained are immediately optionally enriched in vitro. Cell populations can be enriched by positive or negative selection, as is well known in the art. Additionally, the T cells are optionally frozen or frozen and then thawed at a later date.

Before or after introducing the T cell antigen coupler into the T cells, the T cells are optionally activated and/or expanded using methods well known in the art. For example, T cells can be expanded by contacting the surface with bound agents that stimulate CD3/TCR complex-associated signals and ligands that stimulate costimulator molecules on the T cell surface.

Methods of transducing or transfecting a nucleic acid sequence into a T cell and causing the T cell to express the transduced nucleic acid are well known in the art. For example, nucleic acids can be introduced into cells by physical, chemical or biological means. Physical means include, but are not limited to (microinjection, electroporation, particle bombardment, lipofection and calcium phosphate precipitation). Biological means include the use of DNA and RNA vectors.

In one embodiment, viral vectors, including retroviral vectors, are used to introduce and express nucleic acids in T cells. Viral vectors include vectors derived from lentiviruses, murine stem cell virus (MSCV), poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses. The vector optionally contains a promoter driving expression of the transducing nucleic acid molecule in T cells.

Various assays can be used to confirm the presence and/or expression of the transducing nucleic acid sequence and/or the polypeptide encoded by this nucleic acid in T cells. Assays include, but are not limited to, Southern and Northern blotting, RT-PCR and PCR, ELISA and Western blotting.

In one embodiment, T cells expressing the T cell antigen coupler are compared to T cells not expressing the T cell antigen coupler and/or compared to T cells expressing a conventional CAR in the presence of the antigen. T cell activation is enhanced at Enhanced T cell activation can be confirmed by a number of methods, including increased tumor cell line killing, increased cytokine production, increased cytolysis, increased degranulation and/or CD107α, IFNγ, Examples include, but are not limited to, increased expression of activation markers such as IL2 or TNFα. Increases can be measured in individual cells or in cell populations.

As used herein, the term "increased" or "increase" refers to T cells or T cell populations that do not express the T cell antigen coupler, and/or T cells that express conventional CAR or An increase of at least 2%, 5%, 10%, 25%, 50%, 100% or 200% in a T cell or T cell population expressing the T cell antigen coupler as compared to the T cell population ing.

T cells expressing the T cell antigen coupler, optionally autologous T cells, can be administered to a subject in need thereof. Accordingly, T cells transduced and/or expressing a T cell antigen coupler can be formulated as a pharmaceutical composition. Preferably, such T cells are formulated for intravenous administration.

The pharmaceutical composition is prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to a subject, such as combining an effective amount of such T cells with a pharmaceutically acceptable carrier in an admixture. can be done. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences), 20th Edition, Mack Publishing Company, Easton ), Pennsylvania, USA, 2000). On this basis, such compositions include solutions containing these substances together with one or more pharmaceutically acceptable carriers or diluents in a buffer solution having a suitable pH and isotonicity with physiological fluids. are mentioned, but are not limited to these.

Suitable pharmaceutically acceptable carriers include compositions that are chemically inert and non-toxic in nature, which do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Suitable pharmaceutical carriers include, for example, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium hydrochloride (DOTMA), dioleyl Examples include, but are not limited to, sylphosphotidyl-ethanolamine (DOPE) and liposomes. Such compositions should contain a therapeutically effective amount of the compound together with a suitable amount of carrier so as to provide the form for direct administration to the patient.

Pharmaceutical compositions also include, but are not limited to, frozen powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which further include antioxidants, buffers, bacteriostats and such compositions. can contain solutes that render it substantially compatible with the tissue or blood of the intended recipient. Other ingredients that can be present in such compositions include, for example, water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.

(iii) Methods and Uses One aspect of the present disclosure provides the use of trifunctional T cell antigen couplers to target T cells to specific antigens.

Accordingly, also disclosed is the use of modified T cells to treat cancer in a subject in need thereof, wherein the modified T cells comprise a first polynucleotide encoding a target-specific ligand, a TCR complex express a second polynucleotide encoding a body-binding ligand and a third polynucleotide encoding a T-cell receptor signaling domain polypeptide. The present disclosure also relates to a method for treating cancer comprising administering an effective amount of modified T cells to a subject in need thereof. Also disclosed is the use of an effective amount of modified T cells to treat cancer in a subject in need thereof. Further disclosed is the use of the modified T cells in preparing a medicament for treating cancer in a subject in need thereof. Further disclosed are modified T cells for use in treating cancer in a subject in need thereof. In one embodiment, the target-specific ligand binds to an antigen on a cancerous cell, thereby targeting the cancerous cell for delivery of modified T cells.

Cancers that can be treated include any type of neoplastic disease. Examples of cancers that can be treated include breast cancer, lung cancer and leukemia such as mixed lineage leukemia (MLL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia ( ALL (acute lymphoblastic leukemia), but not limited thereto. Other cancers include epithelial malignancies, blastoma, malignant melanoma, non-epithelial malignancies, hematologic cancers, lymphoid malignancies, benign and malignant tumors, and malignancies. This cancer includes non-solid tumors and solid tumors. Cancers that may be treated include tumors that are not or not yet substantially vascularized, and vascularized tumors.

The modified T cells and/or pharmaceutical compositions described herein can be administered or used in living organisms, including humans and animals. The term "subject" as used herein refers to any member of the animal kingdom, preferably a mammal, more preferably a human.

Procedures for isolating, genetically modifying, and administering T cells to a subject in need thereof are known in the art. Specifically, T cells are isolated from a mammal (preferably human), optionally expanded and/or activated as described herein, and transduced with the nucleic acid molecules of the present disclosure. or transfect. The T cells can be autologous to the subject. In another embodiment, the cell can be allogeneic, syngeneic, or xenogeneic to the subject.

The modified T cells can be administered alone or as pharmaceutical compositions, as described herein. Compositions of the present disclosure are preferably formulated for intravenous administration.

Administration of an "effective amount" of modified T cells and/or pharmaceutical composition is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, the effective amount of a given substance may vary depending on the medical condition, age, sex and weight of the individual and the ability of the recombinant protein to elicit the desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, the dose may be divided and administered in several times daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

For example, the modified T cells and/or pharmaceutical compositions described herein may be administered at 10 ⁴ to 10 ⁹ cells per kg body weight, optionally 10 ⁵ to 10 ⁸ cells per kg body weight or A dose of 10 ⁶ to 10 ⁷ cells can be administered. This dose can be administered in single or multiple doses.

Modified T cells and/or pharmaceutical compositions can be administered by any method known in the art including, but not limited to, aerosol inhalation, injection, ingestion, infusion, implantation or implantation. is not limited to The modified T cells and/or pharmaceutical composition may be administered to the subject subcutaneously, intradermally, intratumorally, intranodally, intrathecally or intramuscularly, by intravenous (iv) injection, or intraperitoneally. can be administered to Such modified T cells and/or pharmaceutical compositions can be injected directly into a tumor, lymph node or site of infection.

As used herein and as well understood in the art, "treating" or "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical outcomes include alleviation or amelioration, detectable or undetectable, of one or more symptoms or conditions, reduction in the extent of disease, stabilization of disease (i.e., not exacerbation) ), prevention of disease spread, slowing or slowing disease progression, amelioration or palliation of disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. In one embodiment, "treatment" includes preventing a disease or condition.

The above disclosure generally describes the present application. A better understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present application. Modifications of form and substitution of equivalents are contemplated as circumstances may suggest or provide tactics. Although specific terms have been employed herein, such terms are meant in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the present application.

Example 1
Background and Overview Trifunctional T cell-antigen couplers (Tri-TACs) were developed to better replicate naturally occurring signaling through the TCR while maintaining MHC-unrestricted targeting. Activation of T cells occurs after binding of the TCR and co-receptors (CD4 or CD8) on T cells to MHC and concomitant binding to conserved regions within the MHC molecule (Yin et al., 2012) ( Kuhns and Davis, 2012). Co-receptors are "lipid rafts" (Fragoso et al., 2003), membrane microdomains (He and Marguet, 2008) that are particularly important for the formation of the TCR signaling complex (Arcaro (Arcaro et al., 2000). In addition to ensuring precise microdomain localization of TCR activation complexes, these co-receptors are important protein kinases for T cell activation, Lck (Kim et al., 2003). (Methi et al., 2005) (Acuto and Cantrell, 2000). As previously mentioned, none of the existing chimeric receptors or bifunctional proteins engage co-receptor molecules or Lck. The transmembrane and intracellular regions of the CD4 co-receptor, which localizes to lipid rafts and binds Lck, are fused to a single-chain antibody (UCHT1; SEQ ID NOs: 13 and 14; sequences also in the public domain) that binds CD3. created a new molecule. This construct is designed to bring CD3 molecules and the TCR into the region of lipid rafts, bringing Lck into the vicinity of the TCR, similar to naturally occurring MHC binding. To target this chimeric receptor, designed ankyrin repeats (darpin) were linked to the CD4-UCHT1 chimera to create a trifunctional T cell-antigen coupler (Tri-TAC). In this particular case, darpin was specific for the proto-oncogene erbB-2.

Human T cells were engineered to express prototype Tri-TAC or conventional CAR with the same darpin. As a result, it was revealed that Tri-TAC-manipulated T cells exhibited functionality equivalent to that of conventional CAR in all respects. Interestingly, for two parameters (TNF-α production and CD107a mobilization) Tri-TAC was found to be more active than conventional CAR. Furthermore, the data show that Tri-TAC per molecule shows a significant enhancement of activity. Furthermore, Tri-TAC exhibits improved safety compared to conventional CARs, as the activation domain is not part of this protein.

Containing several signaling domains, conventional CAR is effective in stimulating T cells (Fig. 1C). Incidentally, Tri-TAC (FIG. 1A/B) does not contain any signaling domain of its own. It rests on promoting the proposed interactions between other key players (indicated in gray) in an antigen-dependent manner. To test this design hypothesis, several variants of full-length N-darpin Tri-TAC were generated (Fig. 1D).

Previous studies have demonstrated consistent and significant cell surface expression of CAR molecules. Darpin CAR was found to exhibit strong surface expression (Fig. 2). In contrast, Tri-TAC showed much lower surface expression. This was observed for all mutants with the UCHT1 domain. However, a Tri-TAC mutant lacking the UCHT1 domain showed surface expression similar to darpin CAR.

T cells engineered to express Tri-TAC, Tri-TAC mutants or darpin CAR were stimulated with plate-bound antigen. As a result, T cells engineered to express Tri-TAC and darpin CAR were able to express all the functions measured (TNF-α production, IFN-γ production and CD107a recruitment) (Fig. 3A). and 3B). The binding of Tri-TAC to both CD3 and target antigen was found to be important for T cells to express their function. FIG. 3 shows that removal of UCHT1, which abolishes binding to CD3, abolishes Tri-TAC function. Other data revealed that removal of darpin from Tri-TAC abolished function.

As expected, Tri-TAC-UCHT1-darpin showed no ability to kill antigen-expressing cells when the cytotoxicity of these T cells was examined (Fig. 4). N-darpin Tri-TAC showed a high level of selective cytotoxicity very similar to classical darpin CAR. Interestingly, darpin CAR-expressing T cells appear to exhibit off-target killing at high T cell:target cell ratios (see killing against D2F2 in FIG. 4), whereas Tri-TAC did not show such an effect.

Experimental Examples Figure 1 is a schematic overview. (A) depicts N-darpin Tri-TAC. The Her2-targeting ankyrin repeat domain is fused to the single chain fragment variable (scFv) UCHT1 using a (G ₄ S) ₃ linker. The scFv is then linked to the CD4 molecule. CD4 contains linker and transmembrane regions and a cytoplasmic anchor region. Possible interactions are shown in blurred gray. (B) depicts C-darpin Tri-TAC. In this construct the scFv UCHT1 is replaced with a darpin domain. Again, possible interactions are shown in dim gray. (C) is a model of a classical second generation CAR. The darpin targeting domain is linked to the CD28 transmembrane domain via a CD8α linker. A CD3 zeta domain with three activating ITAM motifs is then bound to the cytoplasmic portion of CD28. (D) Overview of various Tri-TAC controls lacking the darpin targeting domain, the CD3 binding scFv portion or the cytoplasmic portion of the CD4 domain.

FIG. 2 shows the results of phenotypic surface expression analysis of transduced T cells by histogram of each Tri-TAC mutant. T cells were incubated with Her2Fc later detected by flow cytometry. Data presented were gated on CD8+ lymphocytes. The gates shown were chosen relative to untransduced controls.

FIG. 3 shows the results of functional analysis of engineered T cells. In (A), cells were stimulated with plate-bound Her2Fc in medium containing GolgiPlug™ for 4 hours. Cells were first stained for CD8+, then permeabilized and analyzed for TNF-α and IFN-γ production. An initial gate was set for singlet CD8+ lymphocytes. Gates shown were set relative to untransduced controls. In B), cells were stimulated with plate-bound Her2Fc as before. Media contained Golgi Plug™ and anti-CD107a antibody. Actively degranulating cells were expected to have a higher CD107a recycling rate and subsequently show a higher signal of anti-CD107a.

Figure 4 shows cytotoxicity of engineered T cells. Two different adherent mouse tumor lines were plated 24 hours prior to T cell addition. D2F2/E2 has been engineered to express human Her2, but D2F2 has not. Indicated percentages of T cells were added to tumor-containing wells. Tumor cells were incubated with T cells for 6 hours. The T cells were then removed by washing. Media containing 10% Alamar Blue was added to each well for 3 hours. Metabolic activity was determined by endpoint analysis as an index of cell survival. Wells without T cells were defined as maximal viability/metabolic activity and set at 100%, whereas medium incubated without cells was set at 0% metabolic activity. Data presented are the average of three replicate experiments.

DISCUSSION The use of chimeric receptors to redirect T cells to specific targets in an MHC-independent manner is an attractive way to treat cancer and infectious diseases in which pathogen-derived antigens are found on the cytoplasmic membrane. would also be applicable. The chimeric receptor will provide (1) specific cytotoxicity to target cells and (2) minimal off-target toxicity. Conventional CARs are deficient in this regard. This is because such CARs rely on synthetic structures in unnatural positions where the signaling domains may not be properly regulated, thus reducing cellular control of specific activities.

Tri-TAC was designed to redirect the signaling components of the native TCR without ectopic localization of the signaling domain. The design of Tri-TAC was guided by the following principles: (1) chimeric receptors must interact to promote the orderly assembly of key activation protein complexes; It must take advantage of existing cellular adaptations such as the domain environment; and (3) the chimeric receptor must not have any activation domains. Tri-TAC can accomplish this efficiently and, as the data show, with activation kinetics equal to, but not better than, second-generation CARs.

This Tri-TAC is therefore ideally suited for further integration with additional engineered co-receptors to further fine-tune T cell activation. Ultimately, this should result in a substantial reduction of off-target effects without compromising on target cytotoxicity. Tri-TAC appears to be less toxic than existing CARs. Darpin CAR exhibits mild off-target killing at high cell-to-target ratios, which can be problematic when used therapeutically. However, Tri-TAC, while as functional as conventional CAR, showed no off-target effects. Because darpin binds to its target with high affinity, off-target effects on cells expressing high levels of chimeric receptors using darpin are likely. Therefore, without being bound by theory, low surface expression of Tri-TAC would be advantageous as it reduces the likelihood of producing such off-target effects.

Ultimately, the modular nature of Tri-TAC technology allows much finer tuning of the T cell activation process. For example, TCR complex recruitment could be modulated by engineering Tri-TAC molecules with lower CD3 affinity. This could be used to mimic the natural low TCR affinity (Chervin et al., 2009) while retaining a high affinity targeting domain for detecting cancer targets. Unlike classical CAR, the Tri-TAC technology can be manipulated to more closely mimic this.

In conclusion, the presented Tri-TAC technology (1) can efficiently effect T cell activation and cytotoxicity, and (2) does so by mimicking naturally occurring T cell activation. and (3) are highly efficient molecular tools that do not require their own activation domain.

Example 2: Characterization of Tri-TAC technology An overview of the Tri-TAC technology is shown in FIG.

FIG. 5A shows an example of CD8 T cell activation based on co-aggregation of various receptors and their associated partner proteins. First, the major histocompatibility complex I presents the antigen (helix). It is recognized by the T-cell receptor (TCR) complex that can bind to its antigen. This TCR complex contains several individual subunits. Its α/β domains can directly interact with antigens presented on MHC-I. The α/β domains then interact with several other domains (ε, γ, δ and ζ), all of which are involved in T cell activation via various intracellular activation domains. . The TCR complex interacts with MHC-I at the same time as the CD8 co-receptor. This CD8 co-receptor binds MHC-I in an antigen-dependent manner. CD8 directly interacts with Lck, a key protein kinase for activating the TCR receptor complex. We also postulate that the interaction of CD8 with Lck ensures their association with lipid raft (membrane segments) microdomains, which organize and sequester other relevant signaling moieties (dark spheres). It is Later activation leads to recruitment of CD28. If this interaction cascade occurs several times in parallel, T cells can be activated and exert their cytotoxicity.

FIG. 5B shows an overview of chimeric antigen receptors (CARs). CAR seeks to recapitulate the complex mechanisms of T cell activation by combining several key activation domains such as ζ and CD28 into a single synthetic engineered molecule. CAR then uses specific binding domains to directly interact with the antigen of choice. Shown here is an ankyrin repeat protein (darpin). Several such interactions occurring in parallel are thought to lead to T cell activation.

FIG. 5C shows a bispecific T cell engager (BiTE)-like molecule that engages T cells by directly cross-linking the TCR complex to the antigen of choice. The BiTE-like molecule shown here contains two binding domains. The darpin portion interacts with the target antigen. Single chain variable fragment domains (scFv) bind to the TCR complex through their epsilon domains. Several such crosslinks occurring in parallel lead to T cell activation.

FIG. 5D is an overview of TAC technology that mimics the naturally occurring activation process. T-cell antigen couplers (TACs) can bind to their antigens through the darpin-binding domain. Darpin is then linked to an scFv capable of binding to the epsilon domain of the TCR complex. TAC then associates with the CD4 transmembrane and cytoplasmic domains. Like CD8, CD4 interacts with Lck and is located within lipid rafts. Thus, TAC combines TCR recruitment with co-receptor stimulation. Without being bound by theory, it is believed that several such interactions occurring in parallel lead to T cell activation.

Various forms of TAC molecules are possible. FIG. 6A shows a model of a Form 1 TAC molecule. CD4-tail, transmembrane and linker domains bind TCR-epsilon specific scFv (UCHT1). The scFv is then linked to the antigen binding domain. This domain is interchangeable. In this proposal, the antigen binding domain used is a scFv or darpin domain specific for Her2 antigen. FIG. 6B shows Form 2 of the TAC molecule. In this case the CD4 domain first interacts with the antigen binding domain. This domain is then linked to the TCR-recruiting scFv (UCHT1) domain.

Figure 7 shows functionality of scFv CD4 TAC. FIG. 7A is a histogram showing surface expression of scFv CD4 TAC receptor compared to empty vector. Cells were stained with FcHer2 antigen and this antigen was detected with a fluorescently labeled antibody. FIG. 7B shows antigen-specific activation of T cells expressing scFv CD4 TAC (top) or scFv CAR (bottom). T cells expressing scFv CD4 TAC (top) or scFv CAR (bottom) were incubated with plate-bound Her2 antigen. Both modified cells showed antigen-specific activation. A DMSO negative control showed no activity (data not shown). FIG. 7C shows comparable killing of MCF-7 human tumor cell line (Her2 positive) by scFv CD4 TAC and scFv CAR. Both scFv CD4 TAC and scFv CAR were incubated with the MCF-7 human tumor cell line (Her2 positive) and compared to empty vector controls.

FIG. 8 is a characterization of CD4-TAC Form 2. FIG. FIG. 8A is a histogram of darpin CD4-TAC Form 2 in comparison to vector controls. Surface expression was examined using FcHer2 modified antigens. Cells expressing CD4-TAC form 2 show a distinct increase in FcHer2 binding indicative of high surface expression of the receptor. A model of CD4 TAC Form 2 is shown for clarity. FIG. 8B shows Darpin TAC Form 2 engineered T cells exposed to plate-bound Her2 antigen. Cytokine production and degranulation were measured. The data indicate that darpin TAC form 2 is a functional receptor. Treatment without antigen did not activate T cells (data not shown). FIG. 8C shows proliferation of CD4-TAC form 2 compared to empty vector controls. Cells were grown in 100 u/ml IL2 10 ng/ml IL7. Starting with 100,000 cells, proliferation was monitored by counting culture samples at predetermined intervals. Form 2 has a significantly lower growth rate compared to controls.

FIG. 9 shows the functionality of Darpin CD4 TAC Form 1. FIG. FIG. 9A shows surface expression of darpin CD4 TAC (red) in comparison to darpin CAR (green) and NGFR only control (blue). Cells were probed with the receptor specific antigen FcHer2. Histograms show that Darpin CD4 TAC is well expressed on the surface. However, its maximal surface expression is low compared to CAR constructs. FIG. 9B shows proliferation of CD4-TAC form 1. FIG. Proliferation was monitored by sampling and manually counting cells in two weeks of culture. Empty vector shows growth similar to Darpin CAR. However, TAC has reduced proliferation in comparison. Figures 9C and 9D show the percentage of cells positive for various activation and degranulation markers. Empty vector, darpin CD4 and darpin CAR were incubated with plate-bound antigen Her2 or DMSO control. Results from three separate experiments were combined using the statistical analysis software SPICE. Scatter plots show the percentage of cells positive for one set of activation markers. CD4-TAC has a relatively high proportion of degranulation marker-positive cells. Darpin CAR cells are positive for various activation markers, but without a significantly enriched population of degranulation markers. Pie charts show the same data. This indicates that CD4-TAC has a significantly larger population of cells focused on degranulation. CD4-TAC has a large number of activated cells that degranulate with varying levels of cytokine production. However, CAR shows a more random distribution pattern of activation by degranulation, comprising less than 50% of the total population. This pattern would indicate poorly regulated T cell activation by CAR.

Figure 10 shows the cytotoxicity and general activity of TAC and CAR. Cells engineered with TAC, CAR or empty vector controls were incubated with various human tumor cell lines. MDA MB 231, SK OV 3 and A549 all express the Her2 antigen. LOXIMVI is Her2 negative. In all cases, TAC was found to exhibit enhanced cytotoxicity. The antigen-negative cell line LOCIMVI was untargeted, confirming that cytotoxicity is antigen-specific.

FIG. 11 shows receptor surface expression and activation of various TAC controls. FIG. 11A shows cell surface expression (left), degranulation (middle) and cytokine production (right). Constructs lacking specific domains were generated to clarify the significance of such domains. The following domains, the darpin antigen binding domain and the UCHT1 TCR binding domain, have been completely removed, but full-length TAC is shown at the bottom. Surface expression of TACs that do not contain the UCHT1 domain resulted in enhanced surface expression compared to full-length CD4 TACs. Darpin-negative mutants were not detectable using FcHer2 antigen. Degranulation (middle) was observed only in full-length TAC. Deletion of both UCHT1 and darpin did not result in degranulation. Similarly, cytokine production was observed only in full-length TACs. FIG. 11B shows that mouse cell line D2F2 was engineered to express human Her2 antigen (D2F2/E2). Both cell lines were incubated with T cells engineered with full-length CD4-TAC or its deletion mutants. Data indicate an effector to target ratio of 4:1 endpoint. Only full-length CD4-TAC was able to elicit a cytotoxic response. This demonstrates that the darpin and UCHT1 domains are involved in receptor function.

FIG. 12 shows properties of various transmembrane TAC mutants. FIG. 12A is an overview of various such transmembrane constructs. The first set of mutants lack the cytoplasmic domain. CD4 TAC-cytosol has the entire cytoplasmic domain removed. This synthetic construct is replaced by a CD4 TM engineered highly hydrophobic membrane domain. Glycophorin variants replace the CD4 transmembrane domain with the glycophorin transmembrane domain. GPI-anchored variants use CD48 GPI signals arranged to attach TACs to membranes using GPI-anchors. The CD8A TAC mutant replaced the transmembrane and cytoplasmic CD4 domains with their CD8α counterparts. FIG. 12B shows that CD8-purified T cells were engineered with various constructs. Surface expression of various receptors in comparison to full-length TAC is shown. All data are relative to the median fluorescence intensity of controls. All mutants show significantly lower receptor surface expression compared to full-length CD4-TAC. GPI-anchored TAC variants are not detectable above background. FIG. 12C shows the results of examining various mutants for degranulation and cytokine production. Cells were incubated with plate-pounded Her2 antigen. Activity was presented as the percentage of degranulation marker CD107a positive cells (left bar) or all cytokine (TNFa, IFNg and TNFa/IFNg, right bars) producing cells taken together. GPI-fixed or CD8a mutants show background levels of degranulation and cytokine production. Glycophorin, synthetic and -cytosolic TAC variants show moderate levels of degranulation and low levels of cytokine production. In all cases the activity is well below that of full-length CD4-TAC. Taken together, this indicates that anchoring TAC without its cytoplasmic domain results in a less active functional receptor.

FIG. 13 shows the interaction of Lck with TAC mutants. In Figure 13A, the Her2 antigen was covalently attached to magnetic beads. 293TM cells were engineered to express both TAC and TAC cytoplasmic deletion mutants as well as Lck. Beads were incubated overnight with cell lysates, then washed and subjected to Western blotting. Lck was detected using Lck antibody and TAC was detected with Myc antibody. B-actin was used as a control. B-actin was not precipitated and was detected only in the supernatant (S). However, full-length TAC and cytoplasmic deletion were efficiently sedimented and detected in the pellet fraction (B). Vector control and TACs containing no cytoplasmic domain show comparable levels of background Lck signal. However, full-length CD4-TAC shows significant levels of Lck relative to total dose. FIG. 13B shows the results of densitometric analysis of Lck detected in the pellet. Signals were corrected for negative controls. This data confirms that Lck can interact with full-length CD4-TAC.

In FIG. 14 CD4 TAC surface expression and activity are compared with BiTE-like mutants. FIG. 14A shows NGFR-only control (left), CD4-TAC (middle) and BiTE-like mutant (right). Surface expression was examined using transduction markers NGFR and Her2 antigen. TAC shows much lower surface expression compared to BiTE. Most notably, BiTEs appear to secrete sufficient binding antibody to allow transduction-negative cells (NGFR-) to exhibit strong receptor expression. Cytokine production and degranulation are both higher in BiTE-like cells compared to TAC-manipulated cells. Figure 14B compares cytotoxicity in different Her2 positive cell lines (MDA MB 231, SK OV 3, A549). In contrast to cytokine production, TAC-manipulated cells show significantly enhanced cytotoxic activity.

Figure 15 shows a comparison of CD4 TAC WT against a random mutagen library of UCHT1. To examine the ability to alter the properties of TAC, the 24 amino acids present on the binding surface of UCHT1 and TCR epsilon were individually mutated. This theoretically resulted in 480 unique clones, all of which should be represented in this random library. FIG. 15A is a schematic representation of the mutants. Markings indicate mutations that are all at the scFv-epsilon interface. FIG. 5B is a histogram of surface expression. Surface expressed receptors were detected by probing engineered cells with FcHer2 antigen. The library shows considerable enhancement of surface expression of this receptor. FIG. 15C shows WT and library CD4 TAC cells incubated with plate-bound antigen. Their activation and cytokine-producing capabilities have been suggested. This library has similar activity compared to WT. Without being bound by theory, this supports the notion that altering the scFv domain can improve the expression properties of TAC while maintaining its original functional profile.

Figure 16 shows enhanced surface expression of A85V, T161P mutants. Libraries were grown over time to select mutants that showed a growth advantage over WT. A selected mutant (A85V, T161P; numbering is based on the UCHT1 domain fragment) was analyzed. FIG. 16A shows that peripheral blood mononuclear cells (PBMCs) were engineered with WT CD4-TAC or A85V, T161P mutants. Final CD4/CD8 populations were compared between CD4 TAC (left) and A85V, T161P mutant (right). In particular, WT CD4-TAC leads to a reduction in the population of CD4-positive cells. This effect is not observed in mutant cells. FIG. 16B shows surface expression as measured by the NGFR transduction marker and FcHer2 positivity, showing enhanced surface expression of the A85V, T161P mutant. Figure 16C shows that the A85V, T161P mutant has reduced cytokine production (DMSO control showed no activity, data not shown). Degranulation is comparable between WT TAC and A85V, T161P mutants.

Figure 17 shows the cytotoxicity and proliferation of A85V, T161P mutants. In FIG. 17A, WT CD4 TAC and A85V, T161P mutant-engineered T cells were incubated with Her2 antigen-positive cell lines SK OV 3, MDA MB 231 and A549. In all cases the mutants showed reduced levels of cytotoxicity and in the case of A549 no cytotoxicity was detected. In FIG. 17B, cell proliferation was monitored over two weeks in cultures starting with 100,000 cells. Periodically, samples were taken and cells were counted manually. The A85V, T161P mutant shows significantly enhanced growth compared to the WT mutant. Taken together, this indicates that the library is likely to contain a variety of mutants that allow modification and optimization of some TAC functions. Therefore, UCHT1 can be used as a functional regulator.

While this application has been described in terms of what are presently considered to be the preferred embodiments, it should be understood that the application is not intended to be limited to those disclosed embodiments. On the contrary, this application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are referred to to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in its entirety. , which is incorporated herein by reference in its entirety.

References Acuto, O.; and Cantrell, D.M. (2000), "T cell activation and the cytoskeleton", Annual Review of Immunology (Annu. Rev. Immunol.) 18:165-184.
Arcaro, a. , Gregoire, C.I. , Boucheron, N.L. , Stotz, S.; , Palmer, E.P. , Malissen, B. and Luescher, I.M. F. (2000), Essential role of CD8 palmitoylation in CD8 coreceptor function, J. Immunol. 165:2068-2076.
Chames P.S. and Baty, D.M. (2009), "Bispecific antibodies for cancer therapy: the light at the end of the tunnel?", Monoclonal Antibodies ( MAbs) 1:539-547.
Chervin A.; S. , Stone, J.P. D. , Holler, P.M. D. , Bai, A.; , Chen, J.; , Eisen, H.; N. and Kranz, D.; M. (2009), "The impact of TCR-binding properties and antigen presentation format on T cell responsiveness", J. Immunol. 183:1166-1178.
Dotti, G.; , Savoldo, B. , Brenner, M.; (2009), "15 Years of Chimeric Antigen Receptor-Based Gene Therapy: 'Still Close to the Goal? "(Fifteen years of gene therapy based on chimeric antibody receptors: "are we almost there yet?")", Hum. Gene Ther. 20:1229-1239.
Finney, H. M. , Akbar, A.; N. , Lawson, A.; D. G. (2004), Activation of resting human primary T cells with chimeric receptors: co-stimulation from CD28, inducible co-stimulators, CD134 and CD137 contiguous with signals from the TCR zeta chain. T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain)", Journal of Immunology (J.
Fragoso, R. , Ren, D. , Zhang, X. , Su, M.; W. -C. , Burakoff, S.; J. and Jin, Y.; -J. (2003), Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling. and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling,” J. Immunol.
Fry, T.; J. , Mackall, C.I. L. (2013), "T-cell adoptive immunotherapy for acute lymphoblastic leukemia," Hematology American Society Hematology Education Program (Hematology Am. Soc. Hematol. Ed.). .Program) 2013, 348-353.
Han, E. Q. , Li, X. and Han, S.; (2013), "Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges," Journal of Hematology and Oncology. (J. Hematol. Oncol.), 6:47.
He, H.; -T. and Marguet, D.; (2008), T-cell antigen receptor triggering and lipid rafts: issues of space and time scale. Issues regarding the involvement of lipid rafts in T-cell activation. space and time scales. Talking Point on the investment of lipid rafts in T-cell activation.)” EMBO Rep. 9:525-530.
Humphries, C.I. (2013), Adoptive cell therapy: Honing that killer instinct., Nature 504:S13-5.
Kim, P.S. W. , Sun, Z.; J. , Blacklaw, S.; C. , Wagner, G.; and Eck, M.; J. (2003) "A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8," Science 301:1725-1728.
Kochenderfer, J.; N. , Rosenberg, S.; A. (2013), "Treatment B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors," Nature Reviews Clinical Oncology. (Nat. Rev. Clin. Oncol.), 10:267-276.
Kuhns, M.; S. and Davis, M.; M. (2012), "TCR Signaling Emerges from the Sum of Many Parts." Frontiers in Immunol. 3:159.
Methi, T.; , Ngai, J.; , Mahic, M.; , Amarzguioui, M.; , Vang, T. and Tasken, K.; (2005), "Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses," Journal of Immunology. J. Immunol.) 175:7398-7406.
Milone, M.; C. , Fish, J.; D. , Carpenito, C.I. , Carroll, R. G. , Binder, G.; K. , Teachey, D. , Samanta, M.; , Lakhal, M.; , Gross, B. Danet-Desnoyers, G.I. (2009), "Chimeric receptors containing CD137 signal transduction domains mediate enhanced in vivo survival of T cells and enhanced anti-leukemia effects." survival of T cells and increased antileukemia efficacy in vivo).", Molecular Therapy (Mol. Ther.) 17:1453-1464.
Portell, C.; a. , Wenzell, C.I. M. , Advani, A.; S. (2013), "Clinical and pharmacologic aspects of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia", Clinical Pharmacology. Pharmacol.) 5:5-11.
Yin, Y. , Wang, X. X. , and Mariuzza, R.J. a (2012), "Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4", proceedings. National Academy of Sciences (Proc. Natl. Acad. Sci.) U.S.A. S. A. , 109:5405-5410.

Claims (46)

A nucleic acid encoding a single T cell antigen coupler (TAC) polypeptide, said nucleic acid comprising
(a) a first polynucleotide encoding an antigen binding domain that binds to a tumor antigen;
(b) a second polynucleotide encoding a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a third polynucleotide encoding a T-cell co-receptor domain polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said second polynucleotide has at least 90% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 24;
(i) all of components (a), (b) and (c) are directly linked to each other and/or linked by at least one linker to encode a single TAC polypeptide; and
(a), (b) and (c) in the order directly linked to each other, or (a), (b) and (c) in the order, or (b), (a) and (c) in the order linked by at least one linker in
(ii) the nucleic acid does not encode a co-stimulatory domain, and
(iii) binding of antigen to said antigen binding domain of TAC and binding of said ligand to said CD3 protein associated with the TCR complex on T cells promotes activation of said T cells;
nucleic acid. A nucleic acid encoding a single T cell antigen coupler (TAC) polypeptide, said nucleic acid comprising
(a) a first polynucleotide encoding an antigen binding domain that binds to a tumor antigen;
(b) a second polynucleotide encoding a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a third polynucleotide encoding a T-cell co-receptor domain polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said third polynucleotide has at least 90% sequence identity with SEQ ID NO: 17;
(i) all of components (a), (b) and (c) are directly linked to each other and/or linked by at least one linker to encode a single TAC polypeptide; and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the nucleic acid does not encode a co-stimulatory domain, and
(iii) binding of antigen to said antigen binding domain of TAC and binding of said ligand to said CD3 protein associated with the TCR complex on T cells promotes activation of said T cells;
nucleic acid. A nucleic acid encoding a single T cell antigen coupler (TAC) polypeptide, said nucleic acid comprising
(a) a first polynucleotide encoding an antigen binding domain that binds to a tumor antigen;
(b) a second polynucleotide encoding a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a third polynucleotide encoding a T-cell co-receptor domain polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said second polynucleotide has at least 90% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 24;
said third polynucleotide has at least 90% sequence identity with SEQ ID NO: 17;
(i) all of components (a), (b) and (c) are directly linked to each other and/or linked by at least one linker to encode a single TAC polypeptide; and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the nucleic acid does not encode a co-stimulatory domain, and
(iii) binding of antigen to said antigen binding domain of TAC and binding of said ligand to said CD3 protein associated with the TCR complex on T cells promotes activation of said T cells;
nucleic acid. 4. The nucleic acid of any of claims 1-3, wherein the second polynucleotide has at least 99% sequence identity with SEQ ID NO:13 or SEQ ID NO:24. 5. The nucleic acid of claim 4, wherein said second polynucleotide has 100% sequence identity with SEQ ID NO:13 or SEQ ID NO:24. 6. The nucleic acid of any of claims 1-5, wherein the third polynucleotide has 100% sequence identity with SEQ ID NO:17. 7. The nucleic acid of any of claims 1-6, wherein the antigen binding domain binds HER2 or BCMA. 8. The nucleic acid of any of claims 1-7, wherein the first polynucleotide has 100% sequence identity with SEQ ID NO:7. A nucleic acid encoding a single T cell antigen coupler (TAC) polypeptide, said nucleic acid comprising
(a) a first polynucleotide encoding a HER2 antigen binding domain;
(b) a second polynucleotide encoding a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a third polynucleotide encoding a T-cell co-receptor domain polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
(i) all of components (a), (b) and (c) are directly linked to each other and/or linked by at least one linker to encode a single TAC polypeptide; and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the nucleic acid does not encode a co-stimulatory domain; and
(iii) binding of HER2 to said HER2 antigen binding domain of TAC and binding of said ligand to said CD3 protein associated with the TCR complex on T cells promotes activation of said T cells;
nucleic acid. (i) the first polynucleotide has 100% sequence identity with SEQ ID NO:7 or SEQ ID NO:22;
(ii) said second polynucleotide has at least 99% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 24; and/or
(iii) said third polynucleotide has 100% sequence identity with SEQ ID NO: 17;
A nucleic acid according to claim 9 . A nucleic acid encoding a single T cell antigen coupler (TAC) polypeptide, said nucleic acid comprising
(a) a first polynucleotide encoding a BCMA antigen binding domain;
(b) a second polynucleotide encoding a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a third polynucleotide encoding a T-cell co-receptor domain polypeptide comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
(i) all of components (a), (b) and (c) are directly linked to each other and/or linked by at least one linker to encode a single TAC polypeptide; and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the nucleic acid does not encode a co-stimulatory domain, and
(iii) binding of BCMA to the BCMA antigen-binding domain of TAC and binding of the ligand to the CD3 protein associated with the TCR complex on T cells promotes activation of the T cells;
nucleic acid. (i) said second polynucleotide has at least 99% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 24; and/or
12. The nucleic acid of claim 11, wherein (ii) said third polynucleotide has 100% sequence identity with SEQ ID NO:17. 13. The nucleic acid of any of claims 1-12, wherein said nucleic acid does not encode an activation domain. 14. The nucleic acid of any of claims 1-13, wherein said antigen binding domain is an engineered ankyrin repeat (darpin) polypeptide or scFv. 15. The nucleic acid of any of claims 1-14, wherein the ligand that binds to the CD3 protein associated with the TCR complex on T cells is a single chain antibody. 16. The nucleic acid of claim 15, wherein the ligand that binds to the CD3 protein associated with the TCR complex on T cells is UCHT1. 17. The nucleic acid of any of claims 1-16 , wherein (i) component (a) and component (c) are directly linked to component (b). 18. A T cell comprising the nucleic acid of any of claims 1-8 or 13-17. 18. A T cell comprising a nucleic acid according to any one of claims 9, 10 or 13-17. A T cell comprising a nucleic acid according to any of claims 11-17. A T cell antigen coupler (TAC) polypeptide, said polypeptide comprising
(a) an antigen binding domain that binds to a tumor antigen;
(b) a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a T-cell co-receptor domain comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said ligand has at least 90% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 25;
(i) all of components (a), (b) and (c) are directly linked to each other and/or are linked by at least one linker, and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the TAC polypeptide does not have a co-stimulatory domain; and
(iii) binding of antigen to said antigen binding domain of the TAC polypeptide and binding of said ligand to said CD3 protein in association with the TCR complex on T cells promotes activation of said T cells;
Polypeptide. A T cell antigen coupler (TAC) polypeptide, said polypeptide comprising
(a) an antigen binding domain that binds to a tumor antigen;
(b) a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a T-cell co-receptor domain comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said T-cell co-receptor domain has at least 90% sequence identity with SEQ ID NO: 18;
(i) all of components (a), (b) and (c) are directly linked to each other and/or are linked by at least one linker, and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the TAC polypeptide does not have a co-stimulatory domain; and
(iii) binding of antigen to said antigen binding domain of the TAC polypeptide and binding of said ligand to said CD3 protein in association with the TCR complex on T cells promotes activation of said T cells;
Polypeptide. A T cell antigen coupler (TAC) polypeptide, said polypeptide comprising
(a) an antigen binding domain that binds to a tumor antigen;
(b) a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a T-cell co-receptor domain comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
said ligand has at least 90% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 25;
said T-cell co-receptor domain has at least 90% sequence identity with SEQ ID NO: 18;
(i) all of components (a), (b) and (c) are directly linked to each other and/or are linked by at least one linker, and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the TAC polypeptide does not have a co-stimulatory domain; and
(iii) binding of antigen to said antigen binding domain of the TAC polypeptide and binding of said ligand to said CD3 protein in association with the TCR complex on T cells promotes activation of said T cells;
Polypeptide. 24. The TAC polypeptide of any of claims 21-23, wherein said ligand that binds to the CD3 protein has at least 99% sequence identity with SEQ ID NO:14 or SEQ ID NO:25. 25. The TAC polypeptide of claim 24, wherein said ligand that binds to CD3 protein has 100% sequence identity with SEQ ID NO:14 or SEQ ID NO:25. 26. The TAC polypeptide of any of claims 21-25, wherein said T cell co-receptor domain has 100% sequence identity with SEQ ID NO:18. 27. The TAC polypeptide of any of claims 21-26, wherein said antigen binding domain binds HER2 or BCMA. 28. The TAC polypeptide of any of claims 21-27, wherein said antigen binding domain has 100% sequence identity with SEQ ID NO:8. A T cell antigen coupler (TAC) polypeptide, said polypeptide comprising
(a) a HER2 antigen binding domain;
(b) a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a T-cell co-receptor domain comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
(i) all of components (a), (b) and (c) are directly linked to each other and/or are linked by at least one linker, and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the TAC polypeptide does not have a co-stimulatory domain; and
(iii) binding of HER2 to said HER2 antigen binding domain of a TAC polypeptide and binding of said ligand to said CD3 protein in association with the TCR complex on T cells promotes activation of said T cells;
Polypeptide. (i) the HER2 antigen binding domain has 100% sequence identity with SEQ ID NO:8 or SEQ ID NO:23;
(ii) the ligand that binds to the CD3 protein has at least 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 25; and/or
(iii) said T-cell co-receptor domain has 100% sequence identity with SEQ ID NO: 18;
30. The TAC polypeptide of claim 29. A T cell antigen coupler (TAC) polypeptide, said polypeptide comprising
(a) a BCMA antigen binding domain;
(b) a ligand that binds to the CD3 protein associated with the TCR complex on T cells;
(c) a T-cell co-receptor domain comprising a CD4 cytoplasmic domain and a CD4 transmembrane domain;
including
(i) all of components (a), (b) and (c) are directly linked to each other and/or are linked by at least one linker, and
(a), (b) and (c) in that order , or (a), (b) and (c) in that order, or (b), (a) and (c) linked by at least one linker in order,
(ii) the TAC polypeptide does not have a co-stimulatory domain; and
(iii) binding of BCMA to said BCMA antigen-binding domain of a TAC polypeptide and binding of said ligand to said CD3 protein in association with the TCR complex on T cells promotes activation of said T cells;
Polypeptide. (i) the ligand that binds to the CD3 protein has at least 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 25; and/or
(ii) said T-cell co-receptor domain has 100% sequence identity with SEQ ID NO: 18;
32. The TAC polypeptide of claim 31. 33. The TAC polypeptide of any of claims 21-32, wherein said TAC polypeptide does not have an activation domain. 34. The TAC polypeptide of any of claims 21-33, wherein said antigen binding domain is an engineered ankyrin repeat (darpin) polypeptide or scFv. 35. The TAC polypeptide of any of claims 21-34, wherein the ligand that binds to the CD3 protein associated with the TCR complex on T cells is UCHT1. 36. The TAC polypeptide of any of claims 21-35, wherein (i) component (a) and component (c) are directly linked to component (b). 37. A T cell comprising the TAC polypeptide of any of claims 21-28 or 33-36. 37. A T cell comprising the TAC polypeptide of any of claims 29, 30 or 33-36. 37. A T cell comprising the TAC polypeptide of any of claims 31-36. 40. A pharmaceutical composition comprising (a) the T cells of any of claims 18-20 or 37-39, and (b) an excipient. 38. Use of T cells according to claims 18 or 37 in the manufacture of a pharmaceutical composition for the treatment of cancer. 42. Use according to claim 41, wherein the cancer is carcinoma, sarcoma, blastoma, lymphoma, or malignant melanoma. 42. Use according to claim 41, wherein the cancer is breast cancer or lung cancer. 42. Use according to claim 41, wherein the cancer is leukemia (MLL), chronic lymphocytic leukemia (CLL), or acute lymphoblastic leukemia (ALL). 39. Use of T cells according to claims 19 or 38 in the manufacture of a pharmaceutical composition for the treatment of HER2 expressing cancers. 40. Use of T cells according to claims 20 or 39 in the manufacture of a pharmaceutical composition for the treatment of BCMA expressing cancers.

Images